Transgenic mouse assay for compounds affecting amyloid protein processing

ABSTRACT

The construction of transgenic animal models for testing potential treatments for Alzheimer&#39;s disease are described. The models are characterized by a greater similarity to the conditions existing in naturally occurring Alzheimer&#39;s disease, based on expression of all three forms of the β-amyloid precursor protein (APP), APP 695 , APP 751 , and APP 770 ), as well as various point mutations based on naturally occurring mutations, such as the London and Indiana familial Alzheimer&#39;s disease (FAD) mutations at amino acid 717, and predicted mutations in the APP gene. The APP gene constructs are prepared using the naturally occurring promoter, as well as inducible promoters such as the mouse metallothionine promoter, which can be regulated by addition of heavy metals such as zinc to the animal&#39;s water or diet, and promoters such as the rat neuron specific enolase promoter, human β actin gene promoter, human platelet derived growth factor B (PDGF-B) chain gene promoter, rat sodium channel gene promoter, mouse myelin basic protein gene promoter, human copper-zinc superoxide dismutase gene promoter, and mammalian POU-domain regulatory gene promoter. The constructs are introduced into animal embryos using standard techniques such as microinjection. Animal cells can be isolated from the transgenic animals or prepared using the same constructs with standard techniques such as lipofection or electroporation. The transgenic animals, or animal cells, are used to screen for compounds altering the pathological course of Alzheimer&#39;s Disease as measured by their effect on the amount and histopathology of APP and β-amyloid peptide in the animals, as well as by behavioral alterations.

BACKGROUND OF THE INVENTION

This is a continuation of U.S. Ser. No. 07/915,469 filed Jul. 16, 1992,now abandoned, which is a continuation-in-part of U.S. patentapplication Ser. No. 07/817,584, filed Jan. 7, 1992, now abandoned,entitled Transgenic Animal Models for Alzheimer's Disease, by SamuelWadsworth, Benjamin Snyder, Vermuri B. Reddy, and Cha-mer Wei.

Transgenic technology is described for the production of animals thatexhibit symptoms of human Alzheimer's disease through the expression ofthe Alzheimer's precursor protein or a modified version thereof.

Alzheimer's Disease (AD) is a degenerative disorder of the brain firstdescribed by Alios Alzheimer in 1907 after examining one of his patientswho suffered drastic reduction in cognitive abilities and hadgeneralized dementia ("The early story of Alzheimer's Disease", editedby Bick K, Amaducci L, and Pepeu G. (Raven Press, New York 1987). It isthe leading cause of dementia in elderly persons. AD patients haveincreased problems with memory loss and intellectual functions whichprogress to the point where they cannot function as normal individuals.With the loss of intellectual skills the patients exhibit personalitychanges, socially inappropriate actions and schizophrenia ("A guide tothe understanding of Alzheimer's Disease and related disorders", editedby Jorm AF.; (New York University Press, New York 1987). AD isdevastating for both victims and their families, for there is noeffective palliative or preventive treatment for the inevitableneurodegeneration. The most common problems in the Alzheimer's patientare inability to dress unaided, restlessness by day, urinaryincontinence and sleep disturbances. The family members reportembarrassment, anxiety, depression, and a decreased social life.

The impact of AD on society and on the national economy is enormous. Itis expected that the demented elderly population in the United Stateswill increase by 41% by the year 2000. It is expensive for the healthcare systems that must provide institutional and ancillary care for thepatients at an estimated annual cost of $40 billion (Jorm, 1987; Fisher,LM: New York Times, Aug. 23, 1989 D1 "Alzheimer's Disease", edited byReisberg, B.; (The Free Press, New York & London 1983). These factorsimply preventive action must be taken to decrease AD incidence byallocating resources into AD research.

At a macroscopic level, the brains of AD patients are usually smaller,sometimes weighing less than 1,000 grams. At a microscopic level, thehistopathological symptoms of AD include neurofibrillary tangles (NFT),neuritic plagues, and degeneration of neurons. AD patients exhibitdegeneration of nerve cells in the frontal and temporal cortex of thecerebral cortex, pyramidal neurons of hippocampus, neurons in themedial, medial central, and cortical nuclei of the amygdala,noradrenergic neurons in the locus coeruleus, and the neurons in thebasal forebrain cholinergic system. Loss of neurons in the cholinergicsystem leads to a consistent deficit in cholinergic presynaptic markersin AD (Reisberg, 1983; "Alzheimer's Disease and related disorders,research and development" edited by Kelly WE; (Charles C. Thomas,Springfield, Ill. 1984).

AD is associated with neuritic plaques measuring up to 200 μm indiameter in the cortex, hippocampus, subiculum, hippocampal gyrus, andamygdala. One of the principal constituents of neuritic plaques isamyloid, which is stained bycongo red (Reisberg, 1983; Kelly, 1984).Amyloid plaques are extracellular, pink--or rust--colored in brightfield, and birefringent in polarized light. The plagues are composed ofpolypeptide fibrils and are often present around blood vessels, reducingblood supply to various neurons in the brain.

Various factors such as genetic predisposition, infectious agents,toxins, metals, and head trauma have all been suggested as possiblemechanisms of AD neuropathy. However, available evidence stronglyindicates two distinct types of genetic predisposition for AD. First,molecular analysis has provided evidence for mutations in the amyloidprecursor protein (APP) gene in certain AD-stricken families (Goate, etal. Nature 349:704-706 (1991); Murrell, J, et al. Science 254; 97-99,1991; Chartier-Harlin, M-C, et al. Nature 353, 844-846 (1991)). Second,in certain other families with a clear genetic predisposition to AD, themutation maps to chromosome 21 but is distinct from the APP locus(Tanzi, R. E., et al. Nature, 331;528-530 (1988)).

Amyloid plaques are abundantly present in AD patients and in Down'sSyndrome individuals surviving to the age of 40. The plaques are alsopresent in the normal aging brain, although at a lower number. Theseplaques are made up of the amyloid β peptide (β peptide) (Glenner andWong, et al., Biochem. Biophys. Res. Comm. 120:885-890 (1984)), which isalso the main protein constituent in cerebrovascular deposits andneurofibrillary tangles. The peptide is a filamentous material that isarranged in beta-pleated sheets and has a molecular weight of 4.2-4.5kd. It is a hydrophobic peptide comprising 39-42 amino acids. Thedetermination of its amino acid sequence led to the cloning of the APPcDNA (Kang, et al., Nature 325:733-735 (1987); Goldgaber, et al.,Science 235:877-880 (1987); Robakis et al., Proc. Natl. Acad. Sci.84:4190-4194 (1987); Tanzi, et al., Nature 331:528-530 (1988) andgenomic APP DNA (Lemaire et al., Nucl. Acids Res. 17:517-522 (1989);Yoshikai, et al., Gene 87, 257-263 (1990). Three forms of APP cDNAs(APP695, APP751, and APP770) have been isolated, and arise from a singleprecursor RNA by alternate splicing. The gene spans more than 175 Kbwith 18 exons (Yoshikai, et al., 1990). APP contains three extracellulardomains, a transmembrane region and a cytoplasmic domain. The B peptideconsists of 28 amino acids just outside the membrane and 14 residues ofthe hydrophobic transmembrane domain. Thus, the β peptide is a cleavageproduct of APP normally found in brain and other tissues such as heart,kidney and spleen. β peptide deposits, however, are usually found onlyin the brain, although Joachim et al., Nature 341:226-228 (1989) havereported S peptide deposits outside the brain in the skin, intestine,and subcutaneous tissues of most AD patients.

The larger alternate forms of APP (APP751, APP770) consist of all ofAPP695 plus one or two additional domains. APP751 consists of all ofAPP695 plus an additional 56 amino acids which has homology to theKunitz family of serine protease inhibitors (KP1) (Tanzi et al., 1988;Weidemann, et al., Cell 57:115-126 (1989); Kitaguchi, et al., Nature331:530-532 (1988); Tanzi et al., Nature 329, 156 (1987). APP770contains APP751 and an additional 19 amino acid domain homologous to theneuron cell surface antigen OX-2 (Weidemann, et al., Cell 57:115-126(1989); Kitaguchi et al., 1988). APP is post-translationally modified bythe removal of the leader sequence and by the addition of sulfate andsugar groups.

Van Broeckhaven, et al., Science 248:1120-1122 (1990) have demonstratedthat the APP gene is tightly linked to hereditary cerebral hemorrhagewith amyloidosis (HCHWA-D) in two Dutch families. This was confirmed bythe finding of a point mutation in the APP coding region in two Dutchpatients (Levy et al., Science 248:1124-1128 (1990). The mutationsubstituted a glutamine for glutamic acid at position 22 of the βpeptide (position 618 of APP695). In addition, certain families aregenetically predisposed to Alzheimer's disease, a condition referred toas familial Alzheimer's disease (FAD), through mutations resulting in anamino acid replacement at position 717 of the full length protein(Goate, et al., (1991); Murrell et al., 1991; Chartier-Harlin et al.,1991). These mutations co-segregate with the disease within the familiesand are absent in families with late-onset AD.

There are no proven animal models to study AD, although aging nonhumanprimates seem to develop amyloid plaques of β peptide in brainparenchyma and in the walls of some meningeal and cortical vessels.Although aged primates and canines can serve as animal models, they areexpensive to maintain and need lengthy study periods. There are nospontaneous animal mutations with sufficient similarities to AD to beuseful as experimental models. Various models have been proposed inwhich some AD-like symptoms may be induced by electrolysis,transplantation of AD brain samples, aluminum chloride, kainic acid orcholine analogs (Kisner, et al., Neurobiol. Aging 7;287-292 (1986);Mistry, J. S., et al., J Med Chem 29;337-343 (1986)). Flood, et al.(Proc. Natl. Acad. Sci. 88:3363 -3366 (1986), reported amnestic effectsin mice of four synthetic peptides homologous to the β peptide. Becausenone of these share with AD either common symptoms, biochemistry orpathogenesis, they are not likely to yield much useful information onetiology or treatment.

Transgenic mice with the human APP promoter linked to E. coliβ-galactosidase (Wirak, D. O., et al., The EMBO J 10;289-296 (1991)) aswell as transgenic mice expressing the human APP751 cDNA (Quon, D, etal. Nature 352, 239-241 (1991)) or subfragment of the cDNA including theβ peptide (Wirak, D. O., et al., Science 253, 323-325 (1991); Sandhu, F.A., et al., J. Biol. Chem. 266, 21331-21334 (1991); Kawabata, S. Nature354, 476-478 (1991)) have been produced. Results obtained in thedifferent studies appear to depend upon the source of promoter and theprotein coding sequence used. For example, Wirak, et al. (1991) foundthat in transgenic mice expressing a form of the β peptide,intracellular deposits of "amyloid-like" material, reactive withantibodies prepared against APP were observed but did not find otherhistopathological disease symptoms. The intracellular nature of theantibody-reactive material and the lack of other symptoms suggest thatthis particular transgenic animal is not a faithful model system forAlzheimer's disease. Kawabata et al. (1991) report the production ofamyloid plaques, neurofibrillary tangles, and neuronal cell death intheir transgenic animals. In each of these studies, the same peptidefragment, the β peptide plus the 56 remaining C terminal amino acids ofAPP, was expressed. Wirak et al. (1991) used the human APP promoterwhile Kawabata, et al. (1991) used the human thy-1 promoter. Intransgenic mice expressing the APP751 cDNA from the neuron-specificenolase promoter of Quon, D., et al., Nature 352, 239-241 (1991),extracellular deposits of material reactive with antibody preparedagainst APP were observed. What was not shown was whether the depositscontained full-length APP751 or β peptide or both, thus precluding anycorrelation of the deposits with those present in Alzheimer's disease.Quon et al. (1991) also state that the protein encoded by the APP695cDNA expressed from the neuron-specific enolase promoter, does not formextracellular immunoreactive deposits. These results raise thepossibility that although the β peptide is included within the APP695precursor, use of the neuron-specific enolase promoter in conjunctionwith the APP695 cDNA may not present an effective Alzheimer's diseasemodel. Furthermore, the presence of APP immunoreactive deposits is notcorrelated with the age or gene dosage in their particular transgenicmodel.

Alzheimer's disease is a complex syndrome involving pathological andbehavioral aspects. A useful disease model should take thesecomplexities into account. There are multiple proteins expressed fromthe gene with certain forms predominating in a given tissue. In thebrain, the 695 form is predominant, but the mRNAs for additional formsare also present (Golde et al., Neuron 4; 253-267 (1990)). It is notknown whether the ratio of the different forms changes with the age ofthe individual. The various protein forms result from alternativesplicing such that the KI domain and/or the OX-2 domain may or may notbe present in the mature protein. Moreover, the β-peptide results frompost-translational processing of the precursor protein. This process canchange in time as an individual ages, and can be affected by mutationsnot directly affecting the structure of the β-peptide:for example, thefamilial Alzheimer's disease (FAD) mutations at amino acid position 717in the full length protein (Groate, et al., 1991; Murrell, et al., 1991;Chartier-Harlin, et al., 1991). Given these considerations, theproduction of universal animal models for Alzheimer's diseasenecessitates the construction of animal models that take into accountthe effects of known mutations on the phenotype resulting from theexpression of these forms, and the possibility of the ratio of thedifferent forms changing during the lifetime of the animal.

It is therefore an object of the present invention to provide an animalmodel for Alzheimer's disease that is constructed using transgenictechnology.

It is a further object of the present invention to provide transgenicanimals that accurately reflect the expression of different forms of theamyloid precursor protein.

It is a still further object of the present invention to providetransgenic animals characterized by certain genetic abnormalities in theexpression of the amyloid precursor protein.

SUMMARY OF THE INVENTION

The construction of transgenic animal models for testing potentialtreatments for Alzheimer's disease is described. The models arecharacterized by a greater similarity to the conditions existing innaturally occurring Alzheimer's disease, based on the ability to controlexpression of one or more of the three forms of the β-amyloid precursorprotein (APP), APP695, APP751, and APP770, or subfragments thereof, aswell as various point mutations based on naturally occurring mutations,such as the FAD mutations at amino acid 717, and predicted mutations inthe APP gene. The APP gene constructs are prepared using the naturallyoccurring APP promoter of human, mouse, or rat origin, as well asinducible promoters such as the mouse metallothionine promoter, whichcan be regulated by addition of heavy metals such as zinc to theanimal's water or diet. Neuron-specific expression of constructs isachieved by using the rat neuron specific enolase promoter.

The constructs are introduced into animal embryos using standardtechniques such as microinjection or embryonic stem cells. Cell culturebased models can also be prepared by two methods. Cell cultures can beisolated from the transgenic animals or prepared from established cellcultures using the same constructs with standard cell transfectiontechniques.

The specific constructs that are described employ the following proteincoding sequences: the APP770 cDNA; the APP770 cDNA bearing a mutation atamino acid 717; the APP751 cDNA containing the KI protease inhibitordomain without the OX2 domain in the construct; the APP751 cDNA andbearing a mutation at amino acid 717; the APP695 cDNA; the APP695 cDNAbearing a mutation at amino acid 717; the APP leader sequence followedby the β peptide region plus the remaining carboxy terminal 56 aminoacids of APP; the APP leader sequence followed by the β peptide regionplus the remaining carboxy terminal 56 amino acids with the addition ofa mutation at amino acid 717; the APP leader sequence followed by the βpeptide region; the β peptide region plus the remaining carboxy terminal56 amino acids of APP; the β peptide region plus the remaining carboxyterminal 56 amino acids of APP with the addition of a mutation at aminoacid 717; a combination genomic-cDNA APP gene construct; and acombination genomic-cDNA APP gene construct, with the addition of amutation at amino acid 717, operably linked to promoters selected fromthe following: the human APP gene promoter, mouse APP gene promoter, ratAPP gene promoter, metallothionine gene promoter, rat neuron specificenolase gene promoter, human β actin gene promoter, human plateletderived growth factor B (PDGF-B) chain gene promoter, rat sodium channelgene promoter, mouse myelin basic protein gene promoter, humancopper-zinc superoxide dismutase gene promoter, and mammalian POU-domainregulatory gene promoter. Additional constructs include a human yeastartificial chromosome construct controlled by the human APP promoter; ahuman yeast artificial chromosome construct controlled by the human APPpromoter with the addition of a mutation at amino acid 717; theendogenous mouse or rat APP gene modified through the process ofhomologous recombination between the APP gene in a mouse or ratembryonic stem (ES) cell and a vector carrying the human APP cDNA of thewild-type such that sequences in the resident rodent chromosomal APPgene beyond the recombination point (the preferred site forrecombination is within APP exon 9) are replaced by the analogous humansequences; the endogenous mouse or rat APP gene modified through theprocess of homologous recombination between the APP gene in a mouse orrat ES cell and a vector carrying the human APP cDNA bearing a mutationat amino acid position 717 such that sequences in the resident rodentchromosomal APP gene beyond the recombination point (the preferred sitefor recombination is within APP exon 9) are replaced by the analogoushuman sequences bearing a mutation at amino acid 717. These constructscan be introduced into the transgenic animals and then combined bymating of animals expressing the different constructs.

The transgenic animals, or animal cells, are used to screen forcompounds altering the pathological course of Alzheimer's Disease asmeasured by their effect on the amount and histopathology of APP and βpeptide in the animals, as well as by behavioral alterations.

BRIEF DESCRIPTION OF THE DRAWINGS

The boxed portions of the drawings indicate the amino acid codingportions of the constructs. Filled portions indicate the various domainsof the protein as indicated in the Figure Legend. Lines indicatesequences in the clones that are 5' or 3' untranslated sequences,flanking genomic sequences, or introns. The break in the line to theleft of the constructs in FIGS. 7 and 8 indicates the presence of a longDNA sequence.

FIG. 1a is a schematic of the APP 770 cDNA coding sequence.

FIG. 1b is a schematic of the APP770 cDNA coding sequence bearing amutation at position 717.

FIG. 2a is a schematic of the APP751 cDNA coding sequence.

FIG. 2b is a schematic of the APP751 cDNA coding sequence bearing amutation at position 717.

FIG. 3a is a schematic of the APP695 coding sequence.

FIG. 3b is a schematic of the APP695 cDNA coding sequence bearing amutation at position 717.

FIG. 4a is a schematic of a coding sequence for the carboxy terminalportion of APP.

FIG. 4b is a schematic of a coding sequence for the carboxy terminalportion of APP bearing a mutation at position 717.

FIG. 5 is a schematic of a coding sequence for the β peptide portion ofAPP.

FIG. 6a is a schematic of a combination genomic/cDNA coding sequenceallowing alternative splicing of the KI and OX2 exons.

FIG. 6b is a schematic of a combination genomic/cDNA coding sequencebearing a mutation at position 717 and allowing alternative splicing ofthe KI and OX2 exons.

FIG. 7a is a schematic of a human APP YAC coding sequence.

FIG. 7b is a schematic of a human APP YAC coding sequence bearing amutation at position 717.

FIGS. 8a and 8b are a schematic of genetic alteration of the mouse APPgene by homologous recombination between the mouse APP gene in a mouseES cell and a vector carrying the human APP cDNA (either of thewild-type (FIG. 8a) or FAD mutant form) (FIG. 8b) directed to the exon 9portion of the gene. As a result of this recombination event, sequencesin the resident mouse chromosomal APP gene beyond the recombinationpoint in exon 9 are replaced by the analogous human sequences.

DETAILED DESCRIPTION OF THE INVENTION

The constructs and transgenic animals and animal cells are preparedusing the methods and materials described below.

Sources of materials

Restriction endonucleases are obtained from conventional commercialsources such as New England Biolabs (Beverly, Mass.), Promega BiologicalResearch Products (Madison, Wis.), and Stratagene (LaJolla Calif.), etc.Radioactive materials are obtained from conventional commercial sourcessuch as Dupont/NEN or Amersham. Custom-designed oligonucleotides forsite-directed mutagenesis are available from any of several commercialproviders of such materials such as Bio-Synthesis Inc., Lewisville, Tex.Kits for carrying out site-directed mutagenesis are available fromcommercial suppliers such as Promega Biological Research Products,Stratagene, etc. Clones of cDNA including the APP695, APP751, and APP770forms of APP mRNA were obtained directly from Dr. Dmitry Goldgaber, NIH.Libraries of DNA are available from commercial providers such asStratagene, La Jolla, Calif., or Clontech, Palo Alto, Calif. PC12 and3T3 cells were obtained from ATCC (#CRL1721 and #CCL92 respectively). Anadditional PC12 cell line was obtained from Dr. Charles Marotta ofHarvard Medical School, Mass. General Hospital, and McLean Hospital.Standard cell culture media appropriate to the cell line are obtainedfrom conventional commercial sources such as Gibco/BRL. Murine stemcells, strain D3, were obtained from Dr. Rolf Kemler (Doetschman, etal., J. Embryol. Exp. Morphol. 87, 27 (1985)). Lipofectin for DNAtransfection and the drug G418 for selection of stable transformants areavailable from Gibco/BRL.

Isolation of the human APP promoter

A cosmid library, constructed from human placental DNA in the pWE15cosmid vector, was screened by hybridization with a 32p-labeled probeprepared by nick-translation (Maniatis, et al. Molecular Cloning: alaboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. 1989)) of the APP770 cDNA clone. Clones that hybridized with theprobe were picked, purified, and characterized by restriction mapping,hybridization, and DNA sequencing. From one such clone containing a long5' flanking region, a NotI to NruI restriction DNA fragment ofapproximately 25 kb was isolated. This fragment terminates 2 nucleotidesbefore the initiator methionine codon of the Alzheimer's protein-codingregion. This fragment, or a subfragment thereof, is the source of thehuman APP promoter for the constructs described herein. Analogous DNAfragments isolated using the same methods from mouse or rat genomiclibraries are the source of mouse or rat promoters.

Definition of APP cDNA clones

The cDNA clone APP-695 is of the form of cDNA described by Kang, et al.,Nature 325:733-735 ((1987), and represents the most predominant form ofAlzheimer's protein in the brain. The cDNA clone APP-751 is of the formdescribed by Ponte, P, Nature 331, 525-527 (1988). The cDNA cloneAPP-770 is of the form described by Kitaguchi, et al. Nature 331:530-532(1988). This form contains an insert of 225 nucleotides relative to the695 form. The 225 nucleotide insert encodes for the KI domain as well asthe OX-2 domain.

Definition of the APP genomic locus

Characterization of phage and cosmid clones of human genomic DNA cloneslisted in the table below originally established a minimum size of atleast 100 kb for the Alzheimer's gene. There are a total of 18 exons inthe APP gens (Lemaire et al., Nucl. Acid Res, 17;517-522, 1989; Yoshikaiet al., 1990). These results taken together indicate that the minimumsize of the Alzheimer's gene is 175 kb.

    ______________________________________                                        I. Table of Alzheimer's Cosmid and Lambda Clones                                    Name of    Insert                                                       Library                                                                             Clone      Size (Kb)                                                                              Assigned APP Region                                 ______________________________________                                        Cosmid                                                                              1 GPAPP47A 35       25 Kb promoter & 9 Kb intron 1                            2 GPAAP36A 35       12 Kb promoter & 22 Kb intron 1                           3 GAPP30A  30-35    5' coding region                                          4 GAPP43A  30-35    exons 9, 10 and 11                                  Lambda                                                                              1 GAPP6A   12       exon 6                                                    2 GAPP6B   18       exons 4 and 5                                             3 GAPP20A  20       exon 6                                                    4 GAPP20B  17       exons 4 and 5                                             5 GAPP28A  18       exons 4 and 5                                             6 GAPP3A   14       exon 6                                                    7 GAPP4A   19       exon 6                                                    8 GAPP10A  16       exons 9, 10 and 11                                        9 GAPP16A  21       exon 6                                              ______________________________________                                    

Construction of Transgenes

The clones bearing various portions of the human APP gene sequence shownin FIGS. 1-5 are constructed in an analogous manner. First, the polyAaddition signal from SV40 virus as a 253 base pair BclI to BamHIfragment (Reddy et al., Science 200;494-502 (1978) is cloned into amodified vector from the pUC series. Next, the cDNA coding sequences(770, 751, or 695) are inserted. Correct orientation and content of thefragments inserted is determined through restriction endonucleasemapping and limited sequencing.

The clones bearing various carboxy terminal portions of the human APPgene sequence shown in FIGS. 4 and 5 are constructed through severalsteps in addition to those indicated above. First, an APP770 cDNA cloneis digested with Asp718 which cleaves after position 56 (numberingsystem of Kang et al., 1987). The resulting 5' extension is filled inusing the Klenow enzyme (Maniatis et al., 1989) and ligated to ahexanucleotide of the following sequence: AGATCT, the recognition sitefor BglII. After cleavage with BglII, which also cuts after position1769, and re-ligation, the translational reading frame of the protein ispreserved. The truncated protein thus encoded contains the leadersequence, followed by approximately 6 amino acids that precede the βpeptide, followed by the β peptide, and the 56 terminal amino acids ofAPP. The clone in FIG. 5 is created by the introduction through sitedirected mutagenesis of nucleotide 1913 in the clone of FIG. 4a(numbering system of Kang et al., 1987) to a T thus creating atermination codon directly following the last amino acid codon of thepeptide. Each of the APP cDNA sequence clones shown in FIGS. 1-5contains a single NruI site 2 nucleotides upstream from the initiatormethionine codon that is used for attachment of the different promotersused to complete each construct.

Expression clones identical to these but bearing mutations at the aminoacid 717 of the full length protein, the site of the FAD mutation, arealso constructed. Mutations at amino acid 717 are created bysite-directed mutagenesis (Vincent, et al., Genes & Devel. 3, 334-347(1989)) and include mutations of the wild-type val codon to one of thefollowing codons; ile, phe, gly, tyr, leu, ala, pro, trp, met, ser, thr,asn, gln.

The preferred method for construction of the combination cDNA/genomicexpression clones in FIG. 6 is as follows. The TaqI site at position 860(numbering system of Kang, et al., 1987) in an APP770 cDNA clone isconverted to an XhoI site by site-directed mutagenesis. Cleavage of theresulting plasmid with XhoI cuts at the new XhoI site and a pre-existingsite at 930, and releases the KI and OX-2 coding sequence.

The plasmid thus generated serves as the acceptor for the KI and OX-2alternative splicing cassette. The alternative splicing cassette iscreated through a series of cloning steps. First, the TaqI site atposition 860 (numbering system of Kang, et al., 1987) in the genomicclone containing exon 6 and adjacent downstream intron is converted toan XhoI site by site-directed mutagenesis. Cleavage of the resultingplasmid with XhoI cuts at the new XhoI site and an XhoI site within theadjacent intron. This fragment is cloned into the XhoI site in a plasmidvector. Second, the genomic clone containing exon 9 and adjacentupstream intron is cleaved with XhoI (position 930) and cloned into theXhoI site of a plasmid vector. These two junction exon/intron fragmentsare released from their respective plasmid backbones by cleavage withXhoI and either BamHI or BglII, and cloned into the XhoI site of aplasmid vector. The resulting XhoI fragment is cleaved with either BamHIor BglII and the genomic 6.6 kb BamHI segment (Kitaguchi et al., 1988)containing the KI and OX-2 coding region along with their flankingintron sequences are inserted. After cleavage with XhoI, this DNAsegment is inserted into the XhoI site of the modified APP770 cDNAconstructed above. These cloning steps generate a combinationcDNA/genomic expression clone that allows cells in a transgenic animalto regulate the inclusion of the KI and OX-2 domains by a naturalalternative splicing mechanism. An analogous gene bearing a mutation atamino acid 717 is constructed by using the mutated form of APP770 cDNAdescribed above.

Activity of Gene Promoters

Different promoter sequences are used to control expression of APPcoding sequences. The ability to regulate expression of the APP gene intransgenic animals is believed to be useful in evaluating the roles ofthe different APP gene products in AD. The ability to regulateexpression of the APP gene in cultured cells is believed to be useful inevaluating expression and processing of the different APP gene productsand may provide the basis for cell cultured drug screens.

The metallothionine (MT) promoter is well characterized, has beenemployed in transgenic animals, and its expression can be regulatedthrough modulation of zinc and glucocorticoid hormone levels (Palmiteret al., Nature 300, 611-615 (1982)).

The human APP promoter is also characterized with regard to expressionin the CNS (Wirak et al., 1991). It is believed that this promoter isuseful for accurately reproducing temporal and spatial expression ofhuman APP sequences in the CNS of transgenic rodents. In addition to thehuman APP promoter, the APP promoter from mouse and rat is used inconjunction with the various wild-type and mutant APP coding sequences.Although the human APP promoter has been shown to have activity in theappropriate regions of the brain of transgenic mice (Wirak et al.,1991), it is believed that the use of a mouse APP promoter in atransgenic mouse or a rat APP promoter in a transgenic rat will offer aneven more precise pattern of expression in the CNS of transgenicanimals.

As an alternative for the control of human APP expression in neurons,the rat neuron specific enolase gene promoter is used. This promoter hasbeen shown to direct expression of coding sequences in neurons(Forss-Petter et al., Neuron 5;197-197 (1990)).

Other alternatives for use in controlling human APP expression inneurons include the human S actin gene promoter (Ray et al., Genes andDevelopment 5:2265-2273 (1991)), the human platelet derived growthfactor B (PDGF-B) chain gene promoter (Sasahara et al., Cell 64:217-227(1991)), the rat sodium channel gene promoter (Maue et al., Neuron4:223-231 (1990)), the human copper-zinc superoxide dismutase genepromoter (Ceballos-Picot et al., Brain Res. 552:198-214 (1991)), andpromoters for members of the mammalian POU-domain regulatory gene family(Xi et al., Nature 340:35-42 (1989)). The POU-domain is the region ofsimilarity between the four mammalian transcription factors Pit-1,Oct-1, Oct.2, and unc-86, and represents a portion of the DNA-bindingdomain. These promoters are known or believed to result in expressionspecifically within the neurons of transgenic animals.

Expression of human APP in non-neuronal brain cells can be directed bythe promoter for mouse myelin basic protein (Readhead et al., Cell48:703-712 (1987)).

Yeast Artificial Chromosomes

The constructs shown in FIG. 7 are constructed as follows. Largesegments of human genomic DNA, when cloned into certain vectors, can bepropagated as autonomously-replicating units in the yeast cell. Suchvector-borne segments are referred to as yeast artificial chromosomes(YAC; Burke et al. Science 236, 806 (1987)). A human YAC library iscommercially available (Clontech, Palo Alto, Calif.) with an averageinsert size of 250,000 base pairs (range of 180,000 to 500,000 basepairs). A YAC clone of the Alzheimer's gene can be directly isolated byscreening the library with the human APP770 cDNA. The inclusion of allof the essential gene regions in the clone can be confirmed by PCRanalysis.

The YAC-APP clone, shown in FIG. 7a, is established in embryonic stem(ES) cells by selecting for neomycin resistance encoded by the YACvector. ES cells bearing the YAC-APP clone are used to producetransgenic mice by established methods described below under "TransgenicMice" and "Embryonic Stem Cell Methods". The YAC-APP gene bearing amutation at amino acid 717 (FIG. 7b) is produced through the generationof a YAC library using genomic DNA from a person affected by a mutationat amino acid 717. The clone is identified and established in ES cellsas described above.

Genetic Alteration of the Mouse APP Gene

The nucleotide sequence homology between the human and murineAlzheimer's protein genes is approximately 85%. Within thepeptide-coding region, there are three amino acid differences betweenthe two sequences. The val residue that is mutated at amino acid 717 isconserved between mouse, rat, and man. Wild-type rodents do not developAlzheimer's disease nor do they develop deposits or plaques in their CNSanalogous to those present in human Alzheimer's patients. Therefore, itis possible that the human but not the rodent form of B peptide iscapable of causing disease. Homologous recombination (Capecchi, MRScience 244, 1288-1292 (1989)) can be used to convert the mouseAlzheimer's gens in situ to a gens encoding the human B peptide. Thisrecombination is directed to a site downstream from the KI and OX-2domains, for example, within exon 9, so that the natural alternativesplicing mechanisms appropriate to all cells within-the transgenicanimal can be employed in expressing the final gene product.

Both wild-type (FIG. 8, schematic "a") and mutant (FIG. 8, schematic"b") forms of human cDNA are used to produce transgenic modelsexpressing either the wild-type or mutant forms of APP. Therecombination vector is constructed from a human APP cDNA (695 or 770form), either wild-type or mutant at amino acid 717. Cleavage of therecombination vector, for example, at the XhoI site within exon 9,promotes homologous recombination within the directly adjacent sequences(Capecchi, 1989). The endogenous APP gens resulting from this event isnormal up to the point of recombination, within exon 9 in this example,and consists of the human cDNA sequence thereafter.

Mutant Forms of APP Proteins

Expression clones identical to these but bearing mutations at the aminoacid 717 of the full length protein, the site of FAD mutations, are alsoconstructed. Mutations at amino acid 717 are created by site-directedmutagenesis (Vincent, et al., 1989) and include mutations of thewild-type val codon to one of the following codons; ils, phe, gly, tyr,leu, ala, pro, trp, met, ser, thr, ash, gln. Mutations of val-717 toils, phe, and gly, have been described (Goate et al., 1991; Murrell, etal., 1991; Chartier-harlin et al., 1991). None of thesenaturally-occurring mutations are charged or bulky amino acids.Therefore it is believed that replacement of val-717 with the otheramino acids listed may also promote the FAD syndrome and have propertiesthat are useful for animal AD models.

Preparation of Constructs for Transfections and Microlnjections

DNA clones for microinjection are cleaved with appropriate enzymes, suchas Sal1, Not1, etc., and the DNA fragments electrophoresed on 1% agarosegels in TBE buffer (Maniatis et al., 1989). The DNA bands are visualizedby staining with ethidium bromide, excised, and placed in dialysis bagscontaining 0.3M sodium acetate, pH 7.0. DNA is electroeluted into thedialysis bags, extracted with phenol-chloroform (1:1), and precipitatedby two volumes of ethanol. The DNA is redissolved in 1 ml of low saltbuffer (0.2M NaCl, 20 mM Tris™, pH 7.4, and 1 mM EDTA) and purified onan Elutip-D™ column. The column is first primed with 3 ml of high saltbuffer (1M NaCl, 20 mMTris™, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn for three times to bind DNA to the column matrix. After one washwith 3 ml of low salt buffer, the DNA is eluted with 0.4 ml of high saltbuffer and precipitated by two volumes of ethanol. DNA concentrationsare measured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 μg/ml in 5 mMTris™, pH 7.4 and 0.1 mMEDTA. Other methods for purification of DNA formicroinjection are also described in Hogan, et al., Manipulating themouse embryo (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1986), in Palmiter, et al., Nature 300, 611 (1982), in "TheQiagenologist, Application Protocols", 3rd edition, published by Qiagen,Inc., Chatsworth, Calif., and in Maniatis, et al., Molecular Cloning: alaboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. 1989).

Construction of Transgenic Animals Animal Sources

Animals suitable for transgenic experiments can be obtained fromstandard commercial sources such as Charles River (Wilmington, Mass.),Taconic (Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.),etc. Swiss Webster female mice are preferred for embryo retrieval andtransfer. B6D2F₁ males can be used for mating and vasectomized SwissWebster studs can be used to stimulate pseudopregnancy. Vasectomizedmice and rats can be obtained from the supplier.

Microinjection Procedures

The procedures for manipulation of the rodent embryo and formicroinjection of DNA are described in detail in Hogan et al.Manipulating the mouse embryo, Cold Spring Harbor Laboratory, ColdSpring harbor, N.Y. (1986), the teachings of which are incorporatedherein.

Transgenic Mice

Female mice six weeks of age are induced to superovulate with a 5 IUinjection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma)followed 48 hours later by a 5 IU injection (0.1 cc, ip) of humanchorionic gonadotropin (hCG; Sigma). Females are placed with malesimmediately after hCG injection. Twenty-one hours after hCG, the matedfemales are sacrificed by CO₂ asphyxiation or cervical dislocation andembryos are recovered from excised oviducts and placed in Dulbecco'sphosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma).Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml).Pronuclear embryos are then washed and placed in Earle's balanced saltsolution containing 0.5% BSA (EBSS) in a 37.5° C. incubator with ahumidified atmosphere at 5% CO₂, 95% air until the time of injection.

Randomly cycling adult female mice are paired with vasectomized males.Swiss Webster or other comparable strains can be used for this purpose.Recipient females are mated at the same time as donor females. At thetime of embryo transfer, the recipient females are anesthetized with anintraperitoneal injection of 0.015 ml of 2.5% avertin per gram of bodyweight. The oviducts are exposed by a single midline dorsal incision. Anincision is then made through the body wall directly over the oviduct.The ovarian bursa is then torn with watchmakers forceps. Embryos to betransferred are placed in DPBS and in the tip of a transfer pipet (about10-12 embryos). The pipet tip is inserted into the infundibulum and theembryos transferred. After the transfer, the incision is closed by twosutures.

Transgenic Rats

The procedure for generating transgenic rats is similar to that of mice(Hammer et al., Cell 63;1099-112 (1990)). Thirty day-old female rats aregiven a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hourslater each female placed with a proven male. At the same time, 40-80 dayold females are placed in cages with vasectomized males. These willprovide the foster mothers for embryo transfer. The next morning femalesare checked for vaginal plugs. Females who have mated with vasectomizedmales are held aside until the time of transfer. Donor females that havemated are sacrificed (CO₂ asphyxiation) and their oviducts removed,placed in DPBS (Dulbecco's phosphate buffered saline) with 0.5% BSA andthe embryos collected. Cumulus cells surrounding the embryos are removedwith hyaluronidase (1 mg/ml). The embryos are then washed and placed inEBSS (Earle's balanced salt solution) containing 0.5% BSA in a 37.5° C.incubator until the time of microinjection.

Once the embryos are injected, the live embryos are moved to DPBS fortransfer into foster mothers. The foster mothers are anesthetized withketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip). A dorsal midlineincision is made through the skin and the ovary and oviduct are exposedby an incision through the muscle layer directly over the ovary. Theovarian bursa is torn, the embryos are picked up into the transferpipet, and the tip of the transfer pipet is inserted into theinfundibulum. Approximately 10-12 embryos are transferred into each ratoviduct through the infundibulum. The incision is then closed withsutures, and the foster mothers are housed singly.

Embryonic Stem (ES) Cell Methods Introduction of cDNA into ES cells

Methods for the culturing of ES cells and the subsequent production oftransgenic animals, the introduction of DNA into ES cells by a varietyof methods such as electroporation, calcium phosphate/DNA precipitation,and direct injection are described in detail in Teratocarcinomas andembryonic stem cells, a practical approach, ed. E. J. Robertson, (IRLPress 1987), the teachings of which are incorporated herein. Selectionof the desired clone of transgene-containing ES cells is accomplishedthrough one of several means. In cases involving random geneintegration, an APP clone is co-precipitated with a gene encodingneomycin resistance. Transfection is carried out by one of severalmethods described in detail in Lovell-Badge, in Teratocarcinomas andembryonic stem cells, a practical approach, ed. E. J. Robertson, (IRLPress 1987) or in Potter et al Proc. Natl. Acad. Sci. USA 81, 7161(1984). Calcium phosphate/DNA precipitation, direct injection, andelectroporation are the preferred methods. In these procedures, 0.5×10⁶ES cells are plated into tissue culture dishes and transfected with amixture of the linearized APP clone and 1 mg of pSV2neo DNA (Southernand Berg, J. Mol. Appl. Gen. 1:327-341 (1982)) precipitated in thepresence of 50 mg lipofectin in a final volume of 100 μl. The cells arefed with selection medium containing 10% fetal bovine serum in DMEMsupplemented with G418 (between 200 and 500 μg/ml). Colonies of cellsresistant to G418 are isolated using cloning rings and expanded. DNA isextracted from drug resistant clones and Southern blotting experimentsusing an APP770 cDNA probe are used to identify those clones carryingthe APP sequences. In some experiments, PCR methods are used to identifythe clones of interest.

DNA molecules introduced into ES cells can also be integrated into thechromosome through the process of homologous recombination, described byCapecchi, (1989). Direct injection results in a high efficiency ofintegration. Desired clones are identified through PCR of DNA preparedfrom pools of injected ES cells. Positive cells within the pools areidentified by PCR subsequent to cell cloning (Zimmer and Gruss, Nature338, 150-153 (1989). DNA introduction byelectroporation is lessefficient and requires a selection step. Methods for positive selectionof the recombination event (i.e., neo resistance) and dualpositive-negative selection (i.e., neo resistance and gancyclovirresistance) and the subsequent identification of the desired clones byPCR have been described by Joyner et al., Nature 338, 153-156 (1989) andCapecchi, (1989), the teachings of which are incorporated herein.

Embryo Recovery and ES cell Injection

Naturally cycling or superovulated female mice mated with males are usedto harvest embryos for the implantation of ES cells. It is desirable touse the C57B strain for this purpose when using mice. Embryos of theappropriate age are recovered approximately 3.5 days after successfulmating. Mated females are sacrificed by CO₂ asphyxiation or cervicaldislocation and embryos are flushed from excised uterine horns andplaced in Dulbecco's modified essential medium plus 10% calf serum forinjection with ES cells. Approximately 10-20 ES cells are injected intoblastocysts using a glass microneedle with an internal diameter ofapproximately 20 μm.

Transfer of Embryos to Pseudopregnant Females

Randomly cycling adult female mice are paired with vasectomized males.Mouse strains such as Swiss Webster, ICR or others can be used for thispurpose. Recipient females are mated such that they will be at 2.5 to3.5 days post-mating when required for implantation with blastocystscontaining ES cells. At the time of embryo transfer, the recipientfemales are anesthetized with an intraperitoneal injection of 0.015 mlof 2.5% avertin per gram of body weight. The ovaries are exposed bymaking an incision in the body wall directly over the oviduct and theovary and uterus are externalized. A hole is made in the uterine hornwith a 25 gauge needle through which the blastocysts are transferred.After the transfer, the ovary and uterus are pushed back into the bodyand the incision is closed by two sutures. This procedure is repeated onthe opposite side if additional transfers are to be made.

Identification of Transgenic Mice and Rats

Tail samples (1-2 cm) are removed from three week old animals. DNA isprepared and analyzed by both Southern blot and PCR to detect transgenicfounder (F₀) animals and their progeny (F₁ and F₂).

Pathological Studies

The various F₀, F₁, and F₂ animals that carry the microinjectedtransgene are sacrificed by CO₂ asphyxiation and analyzed byimmunohistology for neuritic plaques and neurofibrillary tangles (NFTs)in the brain. Brains of mice and rats from each transgenic line arefixed in 4% paraformaldehyde and sectioned on a cryostat. Sections arestained with antibodies reactive with the APP and/or the β peptide.Secondary antibodies conjugated with fluorescein, rhodamine, horseradish peroxidase, or alkaline phosphatase are used to detect theprimary antibody. These experiments permit identification of amyloidplaques and the regionalization of these plaques to specific areas ofthe brain. Plaques ranging in size from 9 to 50 μm characteristicallyoccur in the brains of AD patients in the cerebral cortex, but also maybe observed in deeper grey matter including the amygdaloid nucleus,corpus striatum and diencephalon. Sections are also stained with otherantibodies diagnostic of Alzheimer's plaques, recognizing antigens suchas Alz-50, tau, A2B5, neurofilaments, neuron-specific enolase, andothers that are characteristic of Alzheimer's plaques (Wolozin, et al.,Science 232, 648 (1986); Hardy and Allsop, Trends in Pharm. Sci. 12,383-388 (1991); Selkoe, Ann. Rev. Neurosci. 12, 463-490 (1989); Arai etal., Proc. Natl. Acad. Sci. USA 87, 2249-2253 (1990); Majocha et al.,Amer. Assoc. Neuropathology Abs; 99,22 (1988); Masters et al., Proc.Natl. Acad. Sci. 82,4245-4249; Majocha et al., Can J Biochem Cell Biol63;577-584 (1985)). Staining with thioflavins and congo red is alsocarried out to analyze co-localization of β peptide deposits withinneuritic plaques and NFTs.

Analysis of APP and β Peptide Expression

mRNA: mRNA is isolated by the acid guanidiniumthiocyanate-phenol:chloroform extraction method (Chomczynski and Sacchi,Anal Biochem 162,156-159 (1987)) from cell lines and tissues oftransgenic animals to determine expression levels by Northern blots.

Protein: APP and β peptide are detected by using polyclonal andmonoclonal antibodies that are specific to the extra-cytoplasmic domain,β peptide region, and C-terminus.

Western Blot Analysis: Protein fractions are isolated from tissuehomogenates and cell lysates and subjected to Western blot analysis asdescribed by Harlow et al., Antibodies: A laboratory manual, (ColdSpring Harbor, N.Y., 1988); Brown et al., J. Neurochem. 40;299-308(1983); and Tate-Ostroff et al, Proc Natl Acad Sci 86;745-749 (1989)).Only a brief description is given below.

The protein fractions are denatured in Laemmli sample buffer andelectrophoresed on SDS-Polyacrylamide gels. The proteins are be thentransferred to nitrocellulose filters by electroblotting. The filtersare blocked, incubated with primary antibodies, and finally reacted withenzyme conjugated secondary antibodies. Subsequent incubation with theappropriate chromogenic substrate reveals the position of APP proteins.

Pathological and Behavioral Studies Pathological Studies

Immunohistology and thioflavin S staining are conducted as describedelsewhere herein.

In situ Hybridizations: Radioactive or enzymatically labeled probes areused to detect mRNA in situ. The probes are degraded approximately to100 nucleotides in length for better penetration of cells. The procedureof Chou et al. J. Psych. Res. 24,27-50 (1990) for fixed and paraffinembedded samples is briefly described below although similar procedurescan be employed with samples sectioned as frozen material. Paraffinslides for in situ hybridization are dewaxed in xylene and rehydrated ina graded series of ethanols and finally rinsed in phosphate bufferedsaline (PBS). The sections are post-fixed in fresh 4% paraformaldehyde.The slides are washed with PBS twice for 5 minutes to removeparaformaldehyde. Then the sections are permeabilized by treatment witha 20 μg/ml proteinase K solution. The sections are re-fixed in 4%parformaldehyde, and basic molecules that could give rise to backgroundprobe binding are acetylated in a 0.1M triethanolamine, 0.3M aceticanhydride solution for 10 minutes. The slides are washed in PBS, thendehydrated in a graded series of ethanols and air dried. Sections arehybridized with antisense probe, using sense probe as a control. Afterappropriate washing, bound radioactive probes are detected byautoradiography or enzymatically labeled probes are detected throughreaction with the appropriate chromogenic substrates.

Behavioral Studies

Behavioral tests designed to assess learning and memory deficits areemployed. An example of such as test is the Morris Water maze (Morris,Learn Motivat. 12;239-260 (1981)). In this procedure, the animal isplaced in a circular pool filled with water, with an escape platformsubmerged just below the surface of the water. A visible marker isplaced on the platform so that the animal can find it by navigatingtoward a proximal visual cue. Alternatively, a more complex form of thetest in which there are no formal cues to mark the platform's locationwill be given to the animals. In this form, the animal must learn theplatform's location relative to distal visual cues.

The procedures applied to test transgenic mice is similar for transgenicrats.

Screening of Compounds for Treatment of Alzheimer's Disease

The transgenic animals and animal cells are used to screen compounds fora potential effect in the treatment of Alzheimer's disease usingstandard methodology. The compound is administered to the animals orintroduced into the culture media over a period of time and in variousdosages, then the animals or animal cells examined for alterations inAPP expression, histopathology, and/or behavior using the proceduresdescribed above.

Example 1: Expression of pMTAPP-1 in NIH3T3 and PC12 Cells

The clone, pMTAPP-1 is an example of the expression vector shown in FIG.1a where the promoter used is the metallothionine promoter. Stable celllines were derived by transfecting NIH3T3 and PC12 cell lines (ATCC#CCL92 and CRL1721). 0.5×10⁶ of NIH3T3 or PC12 cells were plated into100 mm dishes and transfected with a mixture of 5 mg of the Sallfragment and 1 mg of pSV2neo DNA (46) precipitated in the presence of 50mg lipofectin (Gibco, BRL) in a final volume of 100 μl.Polylysine-coated plates were used for PC12 cells, which normally do notadhere well to tissue culture dishes. The cells were fed with selectionmedium containing 10% fetal bovine serum in DMEM or RPMI andsupplemented with G418. Five hundred mg/ml (biological weight) and 250mg/ml of G418 were used to select colonies form NIH3T3 and PC12 cells,respectively. Fifteen days after transfection, colonies of cellsresistant to G418 were isolated by cloning rings and expanded in Tflasks. The presence of APP cDNA in the cells was detected by PCR. usingthe procedure of Mullis and Faloona, Methods Enzymol. 155;335-350(1987), the teachings of which are incorporated herein.

Expression of APP in 25 colonies from each cell line was analyzed byimmunostaining (Majocha etal., 1988). Cells were grown to subconfluenceand fixed in a solution containing 4% paraformaldehyde, 0.12M NaCl, and20 mm Na₃ PO₄, pH 7.0. They were incubated overnight with a primarymonoclonal antibody against a synthetic β peptide sequence (Masters etal., 1985) provided by Dr. Ronald Majocha, Mass. General Hospital,Boston, Mass., followed by a generalized anti-mouse antibody conjugatedto biotin (Jackson ImmunoResearch Labs, Pa.). Immunostaining was thenperformed by adding avidin-horseradish peroxidase (HRP) (Vector Labs,Burlingame, Calif.) and diaminobenzidine as the chromogen (Majocha etal., 1985). The results indicated that the pMTAPP-1 vector wasexpressing APP in both NIH3T3 and PC12 cells.

Example 2: Expression of pEAPP-1 in PC12 Cells

pEAPP-1 is an example of the 25 kb human APP gens promoter linked to andcontrolling expression of a human APP770 cDNA like the construct in FIG.1A. DNA from this construct was transfected into PC12 cells as describedabove. Certain clone of pEAPP-1 transfected cells exhibited adifferentiation phenotype morphologically similar to that exhibited byPC12 cells treated with nerve growth factor (NGF). PC12 cells normallyare fairly round and flat cells. Those transfected with pEAPP-1 havecytoplasmic extensions resembling neurites. PC12 cells treated with NGFextend very long neuritic extensions. Thirteen PC12 cell clonestransfected with pEAPP-1 were selected and propagated. Eight of thesecell clones exhibited the spontaneous differentiation phenotype Withclones 1-8, 1-1, and 1-4 exhibiting the strongest phenotypes. Stainingof pEAPP-1 transfected PC12 cells with antibody against the β peptide asdescribed. above indicated that those cells exhibiting thedifferentiation were also expressing APP. Because PC12 cells transfectedwith the pMTAPP1 clone did not exhibit this phenotype even though theAPP770 cDNA is expressed, these results suggest that expression ofAPP770 from the human promoter has novel properties regarding thephysiology of the cell.

Example 3: Expression of pMTA4 in PC12 Cells

pMTA4 is an example of the type of construct shown in FIG. 4A where thepromoter used is the metallothionine promoter. The protein encoded bythis construct differs slightly from that depicted in FIG. 4. An APP770cDNA clone was digested with Asp718 which cleaves after position 56(number system of Kang, et al., 1987). The resulting 5' extension wasfilled in using the Klenow enzyme (Maniatis). The same DNA preparationwas also cleaved with EcoRI which also cuts after position 1795 and theresulting 5' extension was filled in using the Klenow enzyme (Maniatis).Self-ligation of this molecule results in an expression clone in whichthe truncated protein thus encoded contains the leader sequence,followed by a shortened version of the β peptide starting with thesequence phe-arg-val-gly-ser-of the β peptide followedby the 56 terminalamino acids of APP. DNA from this construct was transfected into PC12cells as described above.

Example 4: Generation of Transgenic Mice expressing APP under thecontrol of the MT-1 promoter.

Transgenic mice were made by microinjecting pMTAPP1 vector DNA intopronuclear embryos. pMTAPP1 is an example of the type of construct shownin FIG. 1a which is operably linked to the metallothionine promoter. Theprocedures for microinjection into mouse embryos are described in"Manipulating the mouse embryo" by B. Hogan et al. (1986). Only a briefdescription of the procedures is described below.

Mice were obtained from Taconic Laboratories (German Town, New York).Swiss Webster female mice were used for embryo retrieval andimplantation. B6D2F₁ males were used for mating and vasectomized Swisswebster studs were used to simulate pseudopregnancy.

Embryo Recovery: Female mice, 4 to 8 weeks of age, were induced tosuperovulate with 5 IU of pregnant mare's serum gonadotropin (PMSG;Sigma) followed 48 hours later by 5 IU of human chorionic gonadotropin(hCG; Sigma). Females were placed with males immediately after hCGinjection. Embryos were recovered from excised oviducts of mated females21 hours after hCG in Dulbecco's phosphate buffered saline with 0.5%bovine serum albumin (BSA; Sigma). Surrounding cumulus cells wereremoved with hyaluronidase (1 mg/ml). Pronuclear embryos were thenwashed and placed in Earle's balanced salt solution containing 0.4% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 7% CO₂,5% O₂, and 88% N₂ until the time of injection.

Microinjection: Elutip-D™ purified Sail DNA was dissolved in 5 mM Tris(pH 7.4) and 0.1 mM EDTA at 3 μg/ml concentration for microinjection.Microneedles and holding pipettes were pulled from Fisher coagulationtubes (Fisher) on a DKI model 720 pipette puller. Holding pipettes werethen broken at approximately 70 μm (O.D.) and fire polished to an I.D.of about 30 ∞m on a Narishige microforge (model MF-83). Pipettes weremounted on Narishige micromanipulators which were attached to a NikonDiaphot microscope. The air-filled injection pipette was filled with DNAsolution through the tip after breaking the tip against the holdingpipette. Embryos, in groups of 30 to 40, were placed in 100l drops ofEBBS under paraffin oil for micromanipulation. An embryo was orientedand held with the holding pipette. The injection pipette was theninserted into the male pronucleus (usually the larger one). If thepipette did not break through the membrane immediately the stage wastapped to assist in penetration. The nucleus was then injected and theinjection was monitored by swelling of the nucleus. Following injection,the group of embryos was placed in EBSS until transfer to recipientfemales.

Transfer: Randomly cycling adult female mice were paired withvasectomized Swiss Webster males. Recipient females were mated at thesame time as donor females. At the time of transfer, the females wereanesthetized with avertin. The oviducts were exposed by a single midlinedorsal incision. An incision was then made through the body walldirectly over the oviduct. The ovarian bursa was then torn with watchmakers forceps. Embryos to be transferred were placed in DPBS and in thetip of a transfer pipet (about 10-12 embryos). The pipet tip wasinserted into the infundibulum and embryos were transferred. After thetransfer, the incision was closed by two sutures.

Analysis Of Mice For Transgene Integration: At three weeks of age tailsamples about 1 cm long were excised for DNA analysis. The tail sampleswere digested by incubating with shaking overnight at 55° C. in thepresence of 0.7 ml 5 mM Tris, pH 8.0, 100 mM EDTA, 0.5% SDS and 350 μgof proteinase K. The digested material was extracted once with an equalvolume of phenol and once with an equal volume of phenol:chloroform (1:1mixture). The supernatants were mixed with 70 μl 3M sodium acetate (pH6.0) and the DNAs were precipitated by adding equal volume of 100%ethanol. The DNAs were spun down in a microfuge, washed once with 70%ethanol, dried and dissolved in 100 μl TE buffer (10 mMtris pH 8.0 and 1mM EDTA).

10-20 μl of DNAs were restricted with BamH1, electrophoresed on 1%agarose gels, blotted onto nitrocellulose paper, and hybridizewith ³²P-labeled APP cDNA fragment. Transgenic animals were identified byautoradiography of the hybridized nitrocellulose filters. The DNAs werealso analyzed by PCR carried out by synthetic primers to generate an 800bp fragment.

A total of 671 pronuclear embryos were microinjected out of which 73live and 6 dead pups were born. DNA analysis identified 9 transgenicmice (5 females and 4 males) which were bred to generate F₁ and F₂transgenics. These animals can be analyzed. for expression of mRNA andprotein of APP in different tissues and for analysis of behavioral andpathological abnormalities as described above.

Modifications and variations of the making and testing of transgenicanimal models for testing of Alzheimer's disease will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe following claims.

We claim:
 1. A method for screening compounds for an effect onAlzheimer's disease comprisingadministering the compound to be tested toa transgenic mouse having integrated into the chromosome a nucleic acidconstruct, wherein the construct comprises a mammalian promoteroperatively linked to a cDNA-genomic DNA hybrid sequence, wherein saidhybrid sequence contains a cDNA sequence encoding APP770 or a cDNAencoding APP770 with a naturally occurring mutation, wherein a genomicAPP DNA sequence consisting of exon 6 and an amount of the adjacentdownstream intron sufficient for Splicing, the KI and OX-2 codingregions and an mount of each of their upstream and downstream intronssufficient for splicing, and exon 9 and an amount of the adjacentupstream intron sufficient for splicing is substituted into the cDNAsequence encoding APP770 or the cDNA sequence encoding APP770 with anaturally occurring mutation, and wherein the construct is transcribedand differentially spliced in the transgenic mouse to form mRNA which istranslated into APP695, APP751 and APP770, and determining if there isaltered expression or processing of APP, wherein the presence of alteredexpression or processing of APP is indicative of an effect of thecompound on Alzheimer's disease.
 2. The method of claim 1 wherein theconstruct includes a mutation at amino acid
 717. 3. The method of claim1 wherein the construct includes a promoter selected from the groupconsisting of the human AlP promoter, metallothionine promoter, ratneuron specific enolase promoter, and human platelet derived growthfactor B chain promoter.
 4. The method of claim 1 wherein the promoteris the human platelet derived growth factor B chain promoter.
 5. Themethod of claim 1 further comprising determining if the brainhistopathology, or the behavior of the transgenic mouse is altered afteradministration of the compound to the mouse.
 6. The method of claim 1wherein altered expression or processing of APP is determined bymeasuring the level of β peptide produced by the transgenic mouse andcomparing the measured level to the level of β peptide produced by atransgenic mouse to which the compound has not been administered.